Role of Src family tyrosine kinases in the down-regulation of epidermal growth factor signaling in PC12 cells.

Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches for various intracellular signaling pathways. SFKs have been implicated in epidermal growth factor (EGF) signaling, although their precise mechanisms of action in this pathway remain elusive. To address this issue, we focused on a membrane microdomain, lipid rafts, where SFKs are enriched. In PC12 cells, the EGF receptor (EGFR) is constitutively concentrated in lipid rafts, and further accumulation takes place upon EGF stimulation, followed by activation of SFKs, especially Src and Yes. Inhibition of SFK or disruption of lipid raft function causes EGF-induced neurite extension of PC12 cells. These effects are accompanied by an extended duration of Erk1/2 activation and are suppressed by a MEK inhibitor. In Csk(-/-) fibroblasts, suppression of SFK results in prolonged EGF-induced activation of Erk1/2, with concomitant suppression of EGFR degradation. Furthermore, analysis of the behavior of labeled EGF in PC12 cells reveals that suppression of SFK activity attenuates the rate of clustering of activated EGFR on the membrane. These results suggest that SFK activity in lipid rafts is required to facilitate the down-regulation of EGF signaling, by regulating the clustering of activated EGFR on the membrane in PC12 cells.